THESIS
2022
1 online resource (xxiii, 353 pages) : illustrations (some color)
Abstract
In the trunk of developing zebrafish embryos, adjacent myotome blocks transmit
contractile force via myoseptal junctions (MJs), dynamic structures that connect the actin
cytoskeleton of skeletal muscle cells to extracellular matrix components via transmembrane
protein complexes in the sarcolemma. I report that the endolysosomal ion channel, two-pore
channel type 1 (TPC1), mediates localized, non-propagating Ca
2+ transients that play a distinct
and required role in the capture and attachment of skeletal muscle cells at the MJs. Use of
antisense morpholinos or CRISPR/Cas9 mutagenesis to disrupt tpcn1 gene expression resulted
in abnormal MJ phenotypes, including skeletal muscle cells detaching from or crossing the
vertical myosepta. I also report that TPC1-decorated endolysosomes are dynam...[
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In the trunk of developing zebrafish embryos, adjacent myotome blocks transmit
contractile force via myoseptal junctions (MJs), dynamic structures that connect the actin
cytoskeleton of skeletal muscle cells to extracellular matrix components via transmembrane
protein complexes in the sarcolemma. I report that the endolysosomal ion channel, two-pore
channel type 1 (TPC1), mediates localized, non-propagating Ca
2+ transients that play a distinct
and required role in the capture and attachment of skeletal muscle cells at the MJs. Use of
antisense morpholinos or CRISPR/Cas9 mutagenesis to disrupt tpcn1 gene expression resulted
in abnormal MJ phenotypes, including skeletal muscle cells detaching from or crossing the
vertical myosepta. I also report that TPC1-decorated endolysosomes are dynamically
associated with the MJs in a microtubule-dependent manner, and that attenuating tpcn1
expression or function disrupted endolysosomal trafficking and resulted in an abnormal
distribution of β-dystroglycan (a key transmembrane component of the dystrophin-associated
protein complex). Together, my data suggest that localized TPC1-generated Ca
2+ signals
facilitate essential endolysosomal trafficking and membrane contact events, which help form
and maintain the MJ following the onset of contractile activity. In a second series of
experiments, I also investigated the possible roles of both TPC1 and two-pore channel type 2
(TPC2) during the formation of the zebrafish notochord, a specialized midline structure that
serves structural and patterning roles during embryogenesis. As notochord vacuoles have
been shown to be lysosome-related organelles, I hypothesized that TPCs might contribute to
their biogenesis. My data suggest isoform specific roles for TPC1 and TPC2 in the development
of the perinotochordal sheath, and the notochord vacuoles, respectively. Taken together, my
results add new knowledge to how TPCs and endolysosomal-generated Ca
2+ signaling
contribute to the regulation of zebrafish myogenesis.
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